Can anyone please tell me the best colorimetric methods for the detection of calcium- ATPases and sodium- potassium ATPases?
Hi Abhilash,
I've been using PiColorLock from Innova Biosciences to measure concentration of free phosphate. They do a kit specifically for ATPases as well.
Here's the link: http://www.innovabiosciences.com/phosphate-detection.html
Hope it helps.
Best regards,
Arthur
A widely used method is the baginsky assay that measures release of free phosphate. There should be plenty of references for this in the litterature and you can easily make all the assay ingredients yourself so you dont have to pay extra for kits etc. The assay can also be carried out in microtitter plates.
Another kit is malachite green which I believe have a little higher detection limit. Alternative assays with very high detection limit includes measuring the release of P32 phosphate from ATP-gamma-32 but then this is not colorimetric.
Hope this helps!
Danny
Hi Abhilash,
indeed, determination of the released inorganic phosphate is widely used to measure the ATP-hydrolysis acitivity of ion pumps. Most assays are based on the methods of Fiske & Subbarow (1925) and Ames (1962). Lanzetta et al. (1979) added the malachite green dye to stabilize the phosphate molybdate complex. By addition or absence of the transported ions, you may distinguish between the Ca2+ and the Na+/K+ ATPase in the same preparation. We are using these assays to measure the H+ ATPase and Ca2+ ATPases in plant cells and it works reliable, is very sensitive, is cheap (no expensive kits!) and can be easily adapted to most experimental conditions. I attached a book chapter (uncorrected proof) in which we included an updated protocol of the assay and you can find the above mentioned references.
Best wishes, Gerhard
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